Evaluation of Sample Preparation Methods for the Analysis of Reef-Building Corals Using 1H-NMR-based Metabolomics

The field of metabolomics generally lacks standardized methods for the preparation of samples prior to analysis. This is especially true for metabolomics of reef-building corals, where the handful of studies that have been published employ a range of sample preparation protocols. The utilization of metabolomics may prove essential in understanding coral biology in the face of increasing environmental threats, and an optimized method for preparing coral samples for metabolomics analysis would aid this cause. The current study evaluates three important steps during samples processing of stony corals: (i) metabolite extraction, (ii) metabolism preservation and (iii) subsampling. Results indicate that a modified Bligh and Dyer extraction is more reproducible across multiple coral species compared to methyl tert-butyl ether and methanol extractions, while a methanol extraction is superior for feature detection. Additionally, few differences are detected between spectra from frozen or lyophilized coral samples. Finally, extraction of entire coral nubbins increases feature detection, but decreases throughput and is more susceptible to subsampling error compared to a novel tissue powder subsampling method. Overall, we recommend the use of a modified Bligh and Dyer extraction, lyophilized samples, and analysis of brushed tissue powder for the preparation of reef-building coral samples for 1H NMR metabolomics.